Biophysical characterization of the interaction partners LOV-HK and PhyR of the Brucella abortus blue-light sensing pathway (Póster)

WANDA M. VALSECCHI; ELIANA S. IBARRA; MARÍA V. COSENTINO; MARÍA L. CERUTTI; FERNANDO A. GOLDBAUM; SEBASTIÁN KLINKE; JIMENA RINALDI

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica (SAB); 2022

Sociedad Argentina de Biofísica (SAB)

Brucella abortus is a Gram-negative intracellular bacterium that affects cattle causing brucellosis, a worldwide disease that can be transmitted to humans. Its genome encodes a sensor histidine kinase (HK) containing a light‑oxygen‑voltage (LOV) domain, LOV‑HK, which plays an important role in the light‑regulated Brucella virulence. Activation of the sensor LOV domain, leads to an increase in autophosphorylation of a histidine residue in the HK domain. Subsequently, the phosphate group is transferred to a conserved aspartate residue in the receiver domain of a response regulator (RR) protein, which elicits an output response and finally modulates gene expression and cellular physiology. Previously we have demonstrated that LOV‑HK functionally interacts with two RRs called PhyR and LovR, constituting a functional two-component system (TCS). Given the fact that PhyR is considered the main RR of this TCS, while LovR may play an auxiliary function, and considering that we have already solved the crystal structure of PhyR, we made different efforts to purify or stabilize the LOV‑HK/PhyR complex in solution with the further aim to obtain its three‑dimensional structure. Here we present results on the biophysical characterization by SLS, DLS and crosslinking, and the efforts to obtain structural information of the complex by direct co‑crystallization. In all these cases, considering the previous experience in the literature with other TCS complexes, we tested the addition of ADP, or an ATP analogue (AMP-PCP) and Mg2+, and BeF3– to mimic the LOV‑HK or PhyR phosphorylation states, respectively.